Techniques that can be used for FRET quantification include:
Acceptor photobleaching: Acceptor photobleaching involves quantifying the rise in donor emission subsequent to photobleaching of the acceptor.
FLIM:FLIM allows researchers to map both the spatial and temporal variations in the lifetime of fluorescent molecules within a single living cell, going beyond decay curves.
Sensitized emission: In sensitized emission, the donor fluorophore is excited, and the resulting signal is captured using emission filters designed to detect both donor and acceptor fluorescence.
Spectral imaging: Spectral imaging involves exciting the sample using one or two specific wavelengths and then capturing the entire spectral profiles of both the donor and acceptor fluorescence.
Fluorescence polarization imaging:Fluorescence polarization imaging involves measuring fluorescence polarization both parallel and perpendicular to the excitation light, ensuring high signal-to-noise ratios.